Project description:Neural-specific mRNA decay measurements by TU-Decay technique in control and Pumilio knockdown embryos These TU-Decay microarrays analyze mRNA levels at three timepoints: a one hour pulse, one hour chase, and three hour chase. Neural-specific RNA purification was achieved using prospero-GAL4 driving UAS-T.g.UPRT. Pumilio knockdown in the nervous system was acheived using UAS-Pum(RNAi) driven by prospero-Gal4.
Project description:Pumilio (PUM) is a Drosophila member of a conserved family of sequence-specific RNA-binding proteins that have been shown to regulate mRNA stability and/or translation in a variety of organisms. PUM has been shown to repress the translation of several mRNAs in the Drosophila early embryo; failure to repress these targets leads to lethal developmental defects. Here we use a combination of microarray-based gene expression profiling and next-generation sequencing to identify more than 200 mRNAs that are associated with full-length PUM protein in early embryos and to define a global role for PUM in mRNA decay. Surprisingly, despite the fact that PUM is maternally supplied and thus is present from the beginning of embryogenesis, the vast majority of PUM-directed decay occurs only after zygotic genome activation. We show that the smaug mRNA, which itself encodes an RNA-binding protein that directs transcript decay, is a direct target of PUM via binding sites in the smg 3'UTR. Whereas the endogenous smaug mRNA and the transgenic reporter mRNA that carries the smaug 3'UTR undergo decay after zygotic genome activation, a reporter with an array of PUM-binding sites decays before zygotic genome activation. These data support a model in which additional cis-elements in the smg 3'UTR delay decay until after zygotic genome activation.
